This program project represents a highly integrated approach to translate basic science findings pertaining to the PTEN (phosphatase and tensin homolog) signaling pathway which is among the most frequently inactivated tumor suppressor gene in human tumors. The overall hypothesis of this proposal is that understanding regulation of PTEN offers unique opportunities for comprehension of novel aspects of PTEN biology for targeting the consequence of its frequent inactivation in human cancer. To achieve this goal, this Program Project unites an internationally renowned group of collaborators who will cross disciplinary boundaries to provide novel insights into PTEN regulation, and to develop new strategies for PTEN-targeted tumor therapy. We propose a comprehensive strategy for covering the PTEN waterfront. For the first time we will define the ubiquitin ligases, proteases, metabolic fluxes, and reactive oxygen species that are linked to the PTEN pathway. We also propose highly focused efforts in the validation of new drug targets, structure-based drug design, and the identification of molecular signatures to indicate patients that will respond to PTEN-targeted therapy. With the integrated support of the four Cores, the five Projects will work together to address the following central questions in PTEN biology: Project 1: Define the mechanisms that control the turnover of PTEN and develop antagonists of these pathways so that PTEN can be stabilized in tumors. Project 2: Determine how PTEN regulates metastasis through the E3 ligase Siah2 and use structure-based drug design to develop antagonists of Siah2 for metastatic disease. Project 3: Define aspects of central carbon metabolism that are regulated by PTEN, and assess whether these metabolic hubs are valid drug targets in PTEN null tumors. Project 4: Use structure-based drug design to develop novel drugs targeting AKT and Siah2. Project 5: Use novel nanosensors to examine oxygen and ROS levels in cultures and animal models of PTEN null tumors. In addition to the administrative Core (A), support will be provided for siRNA constructs and libraries (Core B), analysis of human tumor cell lines and TMAs for molecular signatures of markers identified in the course of the proposed studies (Core C) and for analysis of inhibitors developed against each of the components studied in projects 1-4 in 2D, 3D cultures and animal model (Core D). The availability of nanosensors for monitoring oxygen and ROS offer unprecedented opportunity to assess at high sensitivity the relation between PTEN signaling (and inhibitors developed in this Program Project) ROS and oxygen levels, in culture and animal models. Overall, the combination of molecular biology, biochemistry, metabolomics, structure based drug design and nanotechnology offers a second to none opportunity for integrated studies that address critical unanswered questions in tumor biology centered around key tumor suppressor gene PTEN.